v>EPA
United States
Environmental Protection
Agency
Industrial Environmental Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S8-81 -024 Oct. 1982
Project Summary
IERL-RTP Procedures Manual
Level 1 Environmental
Assessment, Biological Tests
D. J. Brusick and R. R. Young
This document is the second edition
of the IERL-RTP Procedures Manual:
Level 1 Environmental Assessment,
Biological Tests for Pilot Studies. The
first edition, prepared by Battelle-
Columbus Laboratories, was published
in 1977 (EPA-600/7-77-043).
This manual outlines the rationale
and proposed methods for performing
Level 1 health effects and ecological
effects bioassays. The spectra of test
methods contained within both the
preceding categories were changed
somewhat from the first edition. Tests
such as the WI-38 cell toxicity assay
were dropped and other tests (such as
those for insect toxicity, plant root
elongation, and bioaccumulation)
were added. These changes were
instituted to give a more comprehen-
sive testing program.
In addition to those changes in
recommended tests, the second edition
includes new sections on sample
collection and processing documenta-
tion, data evaluation and interpreta-
tion, and quality control/quality
assurance procedures.
This Project Summary was devel-
oped by EPA's Industrial Environ-
mental Research Laboratory. Research
Triangle Park, NC, to announce key
findings of the research project that is
fully documented in a separate report
of the same title (see Project Report
ordering information at back).
Introduction
This bioassay procedures manual is a
guide for studies to be conducted by the
Industrial Environmental Research
Laboratory of the Environmental Pro-
tection Agency (EPA), Research Tri-
angle Park, North Carolina. The manual
is a revision of and supersedes the
"IERL-RTP Procedures Manual: Level 1
Environmental Assessment, Biological
Tests For Pilot Studies," published in
April 19771.
The bioassay procedures in this
manual complement the chemical and
physical procedures of the Level 1
environmental assessment program and
are an integral part of a comprehensive
source assessment strategy. The biolog-
ical testing manual complements the
IERL-RTP Level 1 chemical and physical
procedures manual2. The purpose of
Level 1 is to obtain preliminary informa-
tion, identify problem areas, and provide
the basis for the ranking of streams for
further consideration in the overall
environmental assessment. The recom-
mended biotests for testing the toxicity
and mutagenicity of feed and waste
streams of industrial processes are
each described with a brief summary of
procedures for collecting and preparing
the samples to be tested.
Chapter 1 of the manual defines the
goals and strategy employed in Level 1
testing and gives the background and
philosophy of the phased approach to
environmental assessment.
Chapter 2 discusses the Level 1
sampling activities and pretest-han-
dling procedures that can be used for
most industrial complexes. For each
sample type, the discussion focuses on
the general problem as well as specific
-------
problems of preparation needed for
sampling, the actual sampling proce-
dures, and packaging of samples for
shipment.
Chapters 3 through 5 specify the
Level 1 health effects, aquatic, and
terrestrial bioassay schemes. The
schemes identify the methods of anal-
yses, anticipated output and level of
effort required for implementation, and
the basic format for presenting the
results of the tests.
Chapter 6 describes the data manage-
ment, including data summary forms
and an approach to consolidated toxicity
assessment for multitest data for health
and ecological effects.
Chapter 7 outlines the recommended
quality control, quality assurance, and
documentation procedures necessary
to verify the quality of the assays.
Chapter 8 briefly discusses testing
beyond that defined as Level 1.
Definition of Level 1
Environmental Assessment
Testing
Physical and chemical character-
ization of environmental emissions is
critical to the definition of, need for, and
design of control technology. However,
the final objective of lERL-RTP's environ-
ment assessment is the control of
industrial emissions to meet environ-
mental goals that limit the release of
substances that cause harmful human
health or ecological effects. Conse-
quently, the testing of industrial feed
and waste streams for biological effects
is needed as a complement to the
physical and chemical data to ensure
that the assessment is comprehensive.
Biological testing can provide a direct
measure of the toxicity and/or muta-
genicity of substances to organisms that
chemical analysis cannot. This is
especially important when dealing with
substances for which there is little
available data on toxicity or when
assessing complex mixtures where
synergisms and antagonisms may alter
the toxicity of the individual chemical
constituents.
It should be stressed that the results of
Level 1 tests are not intended for
regulatory actions or recommendations,
nor are they to be used as tests of
acceptability or non-acceptability of
emission release. The three-phased
sampling and testing strategy was
developed to focus available resources
(both manpower and dollars) on indus-
trial emissions which have a high
potential for causing measurable health
or ecological effects and for providing
chemical and biological information on
all sources of industrial emissions.
Classification of Streams For
Sampling Purposes
Comprehensive assessments are
organized around the five general types
of samples found in industrial and
energy-producing processes, rather
than around the analytical procedures
that are required to collect the samples.
The five sample types are:
(1) Gas/Vapor (Non-particulate laden)
— These include samples from
process streams, vents, and ef-
fluents. Samples contain inorgan-
ic and organic gaseous compo-
nents.
(2) Gaseous Streams (Particulate or
aerosol laden) — These involve
sampling contained air or gas
streams such as in ducts or
stacks. Samples include particu-
lates and higher molecular weight
organics with boiling points higher
than 100°C.
(3) Liquid/Slurry Streams — Liquid
streams are defined as those
containing less than 5 percent
solids. Slurry streams are defined
as those containing greater than 5
percent solids. Liquid or slurry
streams are classified as aqueous
or nonaqueous. A stream sample
that contains more than 0.2
percent organics is considered
nonaqueous.
(4) Solids — These include a broad
range of material sizes (from large
lumps to powders and dusts) as
well as nonflowing wet pastes.
Nonflowing wet pastes may be
formed by wetting solids either
with aqueous or nonaqueous
liquids, or they may be highly
viscous liquids such as some tars
or oils. The distinction between
solids and slurries can become
blurred.
(5) Fugitive Emissions — These are
transmitted to the environment
without first passing through some
stack, duct, pipe, or channel
designed to direct or control their
flow. They may be in any of the
above physical forms and may
result from nonducted gaseous,
particulate, or liquid emissions
from the overall plant or process
units.
A flow diagram, showing the overall
relationship of the samples to the Level
1 analysis scheme, is presented in
Figure 1.
The types of samples obtained from
the procedures outlined in Figure 1 are
usually mixtures and present problems
for biological test systems that have
been developed and validated primarily
with pure chemicals. Table 1 summa-
rizes the characteristics of samples
collected from various sources.
All Level 1 bioassays are not suitable
for the entire range of sample types
obtained from industrial sources. Cer-
tain tests, for example, provide reliable
results with solid samples that are
soluble in organic solvent carriers, but
may not be reliable if used to evaluate a
gas or slurry. In other situations, the
amount of sample required for applicable
bioassays is too large to permit the tests
to be performed on the available sample
(such as with SASS* samples).
Suitability of test systems for specific
samples must be judged on an individua
basis; but, as shown in Table 2, sorm
generalizations and recommendation!
can be made with respect to bioassay/
test sample compatibility.
Level 1 Health Effects
Bioassays
The Level 1 health effects tesi
include assays for determining toxici
and mutagenicity at several levels
organisms ranging in complexity fro
bacteria to mammalian cells in cultui
(both permanent cell lines and prima
cells) to intact animals. Table 3 describi
the biological characteristics of tt
target organisms in this group of tesi
The tests are able to detect molecul
changes such as DNA mutation (Am
test), acute cell toxicity (RAM and Ch
tests), and complex toxicological r
sponses in intact animals (WAT test)
Health effects bioassays are used
determine the concentration of te
material that produces either a defin
mutagenic or toxic effect on the t<
organisms in a short period of time. T
Ames Salmonella/'microsome mu
genesis assay (Ames) identifies t
minimum effective concentration (Mi
of a test sample that produces signific<
mutagenesis in any of four tester stra
of Salmonella typhimurium used.
The rabbit alveolar macropha
(RAM) assay measures four endpoi
relating to cell death and metabi
impairment, following 20 hours
* Source Assessment Sampling System, devel
by EPA (IERL-RTP) and manufactured by /
therm Corporation, Mountain View, CA 94C
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Sample for biological analysis
Gases and suspended
paniculate matter
Sorbent
resins
1
Liquids
Solids
Aqueous t 0.2%]
organic^ I
\Organic
With suspended
solids
f
Extract
concentrate
and solvent
exchange
Concentrate
for microbial
and rodent
tests
Solvent
exchange
extracts
Plant stress
ethylene
• Microbial
mutagenesis
• RAM
cytotoxicity
• CHO
cytotoxicity
• Insect
toxicity
• Microbial
mutagenesis
• CHO
cytotoxicity
• Insect
toxicity
Microbial mutagenesis
• CHO cytotoxicity
Rodent acute toxicity
Aquatic toxicity*
Root elongation
• Insect toxicity
Microbial mutagenesis
RAM cytotoxicity
CHO cytotoxicity
• Rodent acute toxicity
• Aquatic toxicity*
Insect toxicity
cu
Sj'
* Aquatic tests include freshwater or marine fish, invertebrate, and algal tests.
Figure 1. Biological analysis overview.
continuous exposure. The effective
concentration of toxicant that reduces
each parameter to 50 percent of the
control (ECso) is calculated. The ECso is
also estimated in the rodent cell (CHO)
clonal toxicity assay based on the
reduction in colony-forming ability of
the cells following 24 hours of continu-
ous exposure. Mortality and physio-
logical observations are recorded in
both the quanta! and quantitative
phases of the acute in vivo test in
rodents (whole animal test, WAT). For
samples exhibiting toxicity in the
quanta! phase, the dose lethal to 50
percent of the animals (LDso) is cal-
culated. Additional health effects end-
points may be measured in modified
versions of the CHO toxicity test.
Results from Level 1 health effects
tests are interpreted by using evaluation
criteria unique to each test. Test
samples are ranked according to relative
mutagenicity or toxicity using guide-
lines presented in the results and data
interpretation section for each test.
Level 1 Aquatic Ecological
Effects Assays
Biological responses and bioaccumu-
lation must be considered, as well as
chemical and physical parameters,
when assessing the potential impact of
complex wastes on the aquatic environ-
ment. Biological testing for aquatic
ecological effects usually consists of
static acute toxicity tests on selected
organisms representative of the various
trophic levels. The bioaccumulation of
components in complex mixtures is
evaluated using a laboratory technique
for simulating bioaccumulation phenom-
mena.
Acute toxicity tests are used to deter-
mine the concentration of test material
that produces an adverse effect on a
specified percentage of the test organ-
isms in a short period of time. Because
mortality is normally an easily detected
and an obviously important adverse
effect, the most common acute toxicity
test is the acute lethality test. The index
most often used with fish is the 96-hour
median lethal concentration (96-hour
LDso), and for macroinvertebrates, the
48-hour effective concentration (48-
hour ECso). The LCso is a statistically
derived estimate of the concentration of
toxicant in dilution water that is lethal to
50 percent of the test organisms during
continuous exposure for a specified
period of time, based on data from one
experiment. This may be supplemented,
in fish tests, with effects on behavior.
The ECso for macroinvertebrates is an
estimate of the concentration of test
material that results in the immobiliza-
tion of 50 percent of the test organisms
during continuous exposure for a
specified period of time in one experi-
ment. Immobilization is defined as lack
of movement except for minor activity of
appendages. This measured effect is
used because death is not always
easily determined with some inverte-
brates.
In algal tests the principal criterion of
toxicity is the effect on growth during
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Table 1. Level 1 Bioassay Sample Requirements
Source Sample
Description
Characteristics
Air
Gas/Vapor (Non-
particulate laden)
Gaseous Streams
(paniculate/aerosol
laden)
Process Fugitive
Emissions
Fugitive Gases
Liquids
All Sources
Solids
Piles. Conveyors,
Bins. etc.
Grab
SASS Cyclone
110 um + 3 um)
SASS • Cyclone
(1 urn)
SASS - Filter
SASS
XAD-2 Resin
High-Volume
Sampler
High-Volume
Sampler
Grab
Grab or
Composite
Grab or
Composite
Gas
So/ids >3 um
Solids 1-3 um
Solids < 1 um
XAD-2 extract
Solids
XAD-2 extract
Gas
Untreated
Untreated solids
Organic, inorganic, or both.
Sample limited by storage capacity.
May be inorganic, organic, or both.
SASS samples may have limited size.
Same as above.
On fiberglass mat. Combine with
SASS 1 -3 um if possible.
Organics in dichloromethane.
Requires solvent exchange
before bioassay.
Organic, inorganic, or both.
Same as SASS XAD-2 Resin.
Same as Grab, above.
Aqueous, nonaqueous, or organic.
Solution, suspension, or slurry.
Unlimited sample except for fugitive
runoff.
Coal, ash, residues, products; organi
and inorganic, unlimited sample.
continuous exposure for a specified
period of time. The exposure period for
the freshwater algal bioassay is 120
hours; that for the marine algal bioassay
is 96 hours. The 96- or 120-hour
effective concentration. (ECso), the
concentration in which algal growth is
inhibited by 50 percent as compared
with growth in the control, is statistically
estimated. For samples which stimulate
algal growth, the stimulatory concen-
tration (SC20) is calculated. The 96- or
120-hour SCao is defined as the concen-
tration causing a stimulation in growth
of 20 percent relative to the control after
96 or 120 hours of exposure. Other
related criteria which may be useful are
the effects on rates of growth, on
maximum standing crops, and on algal
biomass at the end of the assay.
It may aIso be possible to establish the
approximate concentration of test
material which produces no observable
deleterious effect by any of the criteria
under study, which is the No Observed
Effect Concentration (NOEC).
Since the reporting for each test is
unique, evaluation criteria given for
each test in the Level 1 manual are used
to rank the relative toxicity of test
samples in the various tests.
The recommended test organisms in
freshwater tests are the alga Selenas-
trum capricornutum, juvenile fathead
minnow Pimephales promelas, and
early instars of Daphnia magna. The
recommended test period is 120 hours
for the algal test, 96 hours for the fish
study, and 48 hours for the daphnid
study. Thus, the principal finding
obtained from an algal study is the 120-
hour ECso or SC2o; from the fish study,
the 96-hour LCso; and from the daphnid
study, the 48-hour ECso.
The suggested test organisms in
marine tests are the alga Skeletonema
costatum, the juvenile sheepshead
minnow Cypinodon variegatus, and the
adult mysid Mysidopsis bah/a. The
primary parameters of toxicityobtained
from a marine algal study are the 96-
hour ECso or SC2o; from the marine fish
study, the 96-hour LC5o; and from the
mysid study, the 96-hour ECso.
The aquatic tests described in this
section are well suited for Level 1
environmental assessment testin
because they develop useful informatio
quickly and at low cost. The charactei
istics of the freshwater and marin
aquatic ecological effects tests ar
summarized in Table 4. The six recorr
mended aquatic bioassays have bee
routinely used by EPA and others 1
monitor the biological impact of effluen'
on the environment. There alreac
exists a body of published material ar
technical expertise which can assist
the application and interpretation <
Level 1 aquatic testing. These tes
measure the effect of a test material c
organisms that represent three succe
sively more complex trophic leve
characteristic of either fresh or marii
waters. The freshwater or marii
battery of tests is selected, based on tl
type of receiving water into which tl
effluent is discharged. Their princif
limitations are (1) that they usually
not closely simulate the charactensti
of the receiving waters into which t
test effluent is actually being discharge
and (2) that the species tested may t
be representative of the most sensit
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Table 2. Test Sample/Bioassay Compatibilities
Ecological Effects Bioassays3
Health Effects Bioassaysa
1. (
Sample Type
3as/ Vapor {Non-
Ames
Bc
RAM
NC
CHO
NC
WAT
NC
Aquatic
Tests'0
NC
Terrestrial Tests
PSE
R
RE
NC
IT
B
paniculate)
2. Liquids (<5% Solids)
A. Aqueous
B. Nonaqueous*
3. Solids and Slurries
f>5% Solids)
R
R
A
A
R
R
R
A
R
A
B
B
R
A
R
R
A. Soluble
B. Insoluble
C. SASS particulates
R
R
R
A
R
R
R
A
A
R
R
NC
R
R
NC
B
B
NC
R
R
NC
R
R
A
"Standard test abbreviations are:
Ames:Ames Salmonella/m/crosome mutagenesis assay.
RAM: Rabbit alveolar macrophage cytotoxicity assay.
CHO: Rodent Cell clonal toxicity assay.
WA T: Acute in vivo test in rodents (whole animal test).
PSE: Plant stress ethylene test.
RS: Root elongation test.
IT: Insect toxicity assay.
^Aquatic tests include marine or freshwater fish, invertebrate, and algal bioassays.
cIdentification of compatibility abbreviations:
R: Recommended for Level 1 environmental assessment testing.
NC: Sample not compatible with test methodology.
A: Compatible with bioassay with no modifications to protocol. Not recommended for routine Level 1 testing, but may provide
additional information.
B: Compatible with bioassay with modification to protocol. Not recommended for Level 1 testing.
"Nonaqueous liquids include samples with greater than 0.2 percent organics, solvent exchange samples, and sorbent resin extracts.
Extracts must be solvent exchanged to dimethylsulfoxide (DMSO) for testing.
Table3. Characteristics of Level 1 Health Effects Bioassays
Salmonella
Cytotoxicity Assays
Characteristic
Cell Type/ Organ System
Mutagenesis
Prokaryotic Cell-
Enteric Bacteria
Species
RAM
Eukaryotic-Primary
Rabbit Macrophage
Cells
CHO
Eukaryotic-
Hamster
Cell Line
WAT
Integrated Organ
and Tissue Systems
End Point(s) Measured
Point Mutation
Amenable to Sample Types Solids, Liquids,
Particulates
Lethality and
Metabolic
Impairment
Solids, Liquids,
Particulates
Data Expression
Special Features
Positive or Negative ECso (Viability, A TP)
Requires In Vitro
Activation System
to Detect Active
Metabolites
Especially Effective
for Paniculate
Samples Because
Cells are Phagocytic
Cell Lethality
Solids, Liquids,
Particulates
ECso (Clonal)
Detects Effects
on Reproductive
Capacity of Cells.
Same Cells May Be
Used For SCE Assay
Lethality-
Toxic Signs
Solids, Liquids,
Particulates
LDso or Toxic Signs
Can Detect Complex
Toxicological
Phenomena that
are Dependent on
Interactions of
Several Organ
Systems
-------
Table 4. Characteristics of Level 1 Aquatic Ecological Effects Bioassays
Static Acute
Characteristic Aquatic Bioassay
Algal Bioassay
Freshwater Species
Marine Species
End Point(s) Measured
Amenable to Sample Types
Data Expression
Special Features
Fish — Fathead Minnow,
Invertebrate — Daphnia
Fish — Sheepshead Minnow,
Invertebrate — Mysidopsis
Lethality
Liquids, Solids (leachates)
Can detect whole-animal
effects on key aquatic
ecological consumers
Selenastrum
Skeletonema
Cell Population Growth
Liquids, So/ids (leachates)
ECsO, SC2O
Effective measure of
toxicity to aquatic
producers
species native to those waters. They do,
however, make it possible to rank
municipal and/or industrial effluents in
order of relative toxicity.
Level 1 Terrestrial Ecological
Effects Assays
The Level 1 terrestrial ecological
effects tests include assays for deter-
mining toxicity of complex wastes in
plant and insect test organisms. The
tests are able to detect sublethal toxic
response to stress in plants (PSE test),
sublethal and lethal toxic responses in
germinating seeds (RE test), and acute
toxicity and reproductive impairment in
insects (IT test).
These tests provide a range of
terrestrial organisms for assessing the
effect of effluent streams on the
environment. Test organisms include
maturing plants, germinating seeds,
and insects. This group of tests offers
testing capabilities for all sample types
(including gases) with the advantages of
low cost, reproducibility, and relatively
rapid performance time. The character-
istics of the terrestrial ecological effects
bioassays are summarized in Table 5. A
future goal for this manual is to include
a test procedure for assessing the
impact of effluent samples on soil
microorganisms (decomposers).
Terrestrial ecological tests are used to
determine the concentration of test
material that produces a defined toxic
effect on a specified percentage of the
test organisms in a fixed amount of
time. The plant-stress-ethylene (PSE)
test is designed to assess and rank the
toxic effects of gaseous effluents on
plants by measuring the stress ethylene
of plant response and by assessing
relative foliar injury in exposed plants.
The root-elongation (RE) test measures
the inhibition of root elongation and
seed germination. Although both param-
eters are observable toxic responses
and are reported, root-elongation inhi-
bition is the preferred end point. The
concentration which inhibits root elon-
gation by 50 percent of the control (EC5o)
is estimated and used to rank effluent
samples. The insect-toxicity assay
measures the acute toxicity and repro-
ductive capacity of fruit flies treated
with environmental samples. The dose
lethal to 50 percent of the flies (LD50)
compared to the control is calculated
and used to rank test samples. In the
optional fertility test, the effective
concentration which reduces the fe-
cundity of surviving dosed flies to 50
percent of control flies (EC50) is calcu-
lated.
The Level 1 terrestrial tests represent
the state of the art for environmental
assessment for terrestrial ecological
effects. These tests have not been as
thoroughly validated with complex envi-
ronmental mixtures as have the health
and aquatic ecological effects tests.
Level 1 Data Formatting and
Analysis
Procedures are described in the Level 1
manual so that data from all Level 1
bioassays can be organized into a
uniform evaluation format to aid in the
use and interpretation of the data. This
format is structured so that data can be
converted from the conventional bio-
assay output into four levels of response:
Nondetectable (ND), Low (L), Moderate
(M), and High (H).
To ensure uniform data recording and
translation of raw data into the final
summarized form, standard data record-
ing and data transition forms have been
developed 3>". Data transition forms are
used in sequence for data summary and
analysis. The critical data values
determined for each assay (MEC, ECso,
LDso, or LCso) are recorded on critical
data summary forms. Samples of health
effects and aquatic ecological effects
critical data summary forms are pre-
sented. Test materials are then ranked
based on the critical data value;
reported in the summary forms. Evalua
tion criteria are presented for each test
The ranking of the level of response of;
sample in each test is then recorded 01
the bioassay summary table presente<
in the manual.
Level 1 Quality Control and
Quality Assurance
Requirements
If Level 1 assessments are to be use
as a basis for decisions regardm
further bioassay assessment, it will b
necessary to ensure the quality of th
test data. Quality control standardizatio
and standardized data documentatio
will contribute significantly to ensuranc
of test quality and reproducibility.
A separate set of documents outlinir
recommended quality control an
quality assurance procedures has bee
prepared and is available as a guide ft
laboratories conducting Level 1 bii
assays. The procedures described ai
consistent with the intent and requir<
ments of the U.S. Food and Dri
Administration's (FDA's) Good Labor
tory Practice (GLP) Regulations5. The:
guides outline the basic steps involvi
in the Level 1 procedures and provii
sample quality control recording forr
for sample data collecting. The qual
control/quality assurance documer
will be especially helpful to laboraton
beginning to conduct Level 1 testing
-------
completeness based on the suggestions
in both the Level 1 manual and the
quality control and quality assurance
manual.
In order to ensure the quality of test
results from biological laboratories
involved in the environmental assess-
ment program, audit samples have been
made available, either as blind samples
during analysis of assessment samples,
or separately as coded unknown sam-
ples. Coded laboratory quality assurance
samples have been prepared to submit
to laboratories wishing to ensure Level
1 testing proficiency.
In addition to audit samples, the
quality control and quality assurance
manual is also designed to define the
level of documentation required to
comply with the proposed FDA GLP
regulations. From time to time it will be
necessary to review raw data and final
bioassay reports for consistency and
Environmental Assessment
Beyond Level 1
Level 1 environmental assessment
should provide an accurate ranking of
emissions of stationary sources with
respect to their potential toxicity.
Moreover, the ranking should ensure
that the toxicity isf rom the emissions as
released into the environment. Level 1
assessment should also generate
information concerning rate of effluent
discharge into the environment and
environmental fate of the emission.
A composite of summarized bioassay
and chemical analysis data will provide
a measure of toxicity and the potential
for damage to the environment. This
concept is outlined in Figure 2.
Table 5. Characteristics of Level 1 Terrestrial Ecological Effects Bioassays
Characteristic
Plant Stress
Ethylene Test
Root Elongation
Test
Insect Toxicity
Test
Species
End Point(s)
Measured
Amenable to
Sample Types
Data Expression
Special Features
Bush Bean
Metabolic stress
evidenced by
ethylene production
Gases, Liquids
Positive or Negative
Only validated
Level J Bioassay
for gases; sensitive
Cucumber. Wheat,
Red Clover,
Radish, Lettuce
Root length
Liquids, Solids
(leachates)
Detects toxicity
to terrestrial
producers; multiple
species
Orosophila
melanogaster
Lethality, Repro-
ductive capacity
Liquids, Solids
LD50
Detects lethality to
terrestrial consumer
plus can provide
data on fertility
Assessment of
environmental
fate
Measurement and
Emission ^ documentation
site of release date
Bioaccumulation
measurement
Sample collection
(define conditions)
Level 1
. chemical _*J Discharge
analysis* severity
Weighted Quantitative
discharge —»• ranking of
severity fwdsj * toxicity
Pretest
' processing'
* Defined in IERL-RTP Manual:
Level 1 Environmental Assessment (Second Edition) (Reference 2)
Level 1
bioassays
Toxicity
definition
Data
normalization
from pretest
processing
I
Figure 2. Proposed scheme for a second stage evaluation of Level 1 results.
-------
References
1. Duke, K.M., David, M.E., and Dennis,
A.J. "IERL-RTP Procedures Manual:
Level 1 Environmental Assessment,
Biological Tests for Pilot Studies,"
EPA-600/7-77-043 (NTIS PB 268484),
Battelle-Columbus Laboratories,
Columbus, OH, April 1977,114 pp.
2. Lentzen, D.E., et al. "IERL-RTP Pro-
cedures Manual: Level 1 Environ-
mental Assessment (Second Edition),"
EPA-600/7-78-201 (NTIS PB 293795),
Research Triangle Institute, Research
Triangle Park, NC, October 1978,
279 pp.
3. Brusick, D.J. "Level 1 Biological
Testing Assessment and Data For-
matting," EPA-600/7-80-079 (NTIS
PB 80-184914), Litton Bionetics,
Inc., Kensington, MD, April 1980,
100pp.
4. Brusick, D.J., et al. "Procedures for
Quality Control and Quality Assur-
ance of the Level 1 Health Effects
Bioassays," EPA Contract No. 68-
02-2681, Technical Directive 403,
Litton Bionetics, Inc., Kensington,
MD, February 1982. In preparation.
5. DHEW Food and Drug Admmistra
tion. "Nonclinical Laboratory Studies
Good Laboratory Practice Regula
tions," Fed. Regist. Dec. 22, Part I
1978, pp. 59986-60020.
D. J. Brusick and R. R. Young are with Litton Bionetics, Inc., Kensington, MD
20895.
Raymond G. Merrill is the EPA Project Officer (see below).
The complete report, entitled "IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment, Biological Tests," (Order No. PB 82-228 966; Cost:
$13.50. subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Industrial Environmental Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
S8S ?8VIR*2PROTEC1ION AGENCY
CHICAGO It 60604
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